Mutation of the p53 gene is a critical event in the elaboration of many tumors of diverse origin. Alterations in components of the p53 pathway, such as amplification of the mdm2 gene which encodes a p53 inhibitor, have also been found in human tumors. The p53 protein is activated in response to DNA damage and serves a checkpoint function to eliminate or repair cells with damaged DNA. Several upstream signals posttranslationally modify p53 in response to stress. Modifications included phosphorylation at the amino terminus to dislodge the negative inhibitor, MDM2, and the carboxyl terminus to activate the intrinsic DNA binding activity of p53. Due to the large number of phosphorylated amino acids, a combination of modifications may activate p53 and may specify which pathway, growth arrest or apoptosis, to initiate. The first aim is to determine how posttranslational modifications and/or protein-protein interactions regulate the activity of p53 and determining its ability to activate specific targets. Another novel mechanism of activating p53 in response to UV damage appears to be the generation of small MDM2 proteins that can bind full-length MDM2 effectively removing it from the cell. Thus, the second aim will be to examine the ability of altered MDM2 products to activate p53 in response to different DNA-damaging agents and independently of other mechanisms that activate p53. Since loss of mdm2 results in p53-mediated embryo lethality, they have also generated a conditional allele of mdm2 to examine the requirement for mdm2 in dividing and non-dividing cells. In this aim they plan to cross mice expressing a conditional mdm2 allele to mice expressing Cre in cycling thymocytes or in differentiated muscle cells. The experiments proposed in this application will examine critical regulators of p53 such as post translational modifications and interactions with MDM2 in determining p53 activity.